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Peptides, Vol. 9, pp. 945--955. ©PergamonPress plc, 1988. Printedin the U.S.A. 0196-9781/88$3.00 + .00 Endooligopeptidase A Activity in Rabbit Heart: Generation of Enkephalin From Enkephalin Containing Peptides MARIA APARECIDA CICILINI, MARIA JOSE FONSECA RIBEIRO,* EDUARDO BRANDT DE OLIVEIRA,$ RENATO ARRUDA MORTARA't AND ANTONIO CARLOS MARTINS DE CAMARGO§ 1 Department of Physiological Sciences, Universidade Federal do Espirito Santo, ES, Brazil *Department of Toxicology, Faculdade de Ci~ncias FarmacPuticas de Ribeirao Preto Universidade de Sao Paulo, SP, Brazil tDepartment of Parasitology Escola Paulista de Medicina, Sao Paulo, SP, Brazil "~Department of Immunology, and §Department of Pharmacology, lnstituto de Ci~ncias Biom~dicas Universidade de Sao Paulo, Caixa Postal 4365, 01000 Sao Paulo, SP, Brazil R e c e i v e d 13 J a n u a r y 1988 CICILINI, M. A., M. J. F. RIBEIRO, E. B. DE OLIVEIRA, R. A. MORTARA AND A. C. M. DE CAMARGO. Endoo/igopeptidase A activity in rabbit heart: Generationof enkepha/infrom enkephalin containingpeptides. PEPTIDES 9(5) 945--955, 1988.--Two endopeptidases displaying similar specificities towards peptide hormone substrates but differing in molecular size have been identified in rabbit heart and isolated by a combination of ion-exchange chromatography, gel filtration and preparative gel electrophoresis. These two enzymes share several properties with the previously described rabbit brain endooligopeptidase A. They were shown to produce, by a single peptide bond cleavage, [Met5] enkephalin and [LeuS]enkephalinfrom small enkephalin containingpeptides. They also hydrolyze the PheS-Ser5 bond of bradykinin and the Arg~-Arg9 bond of neurotensin. Characteristically, the activity of both low and high Mr enzymes is restricted to oligopeptides. Both forms of heart endooligopeptidase A are inhibited by antibodies raised against the brain enzyme. When electrophoresed in SDS-polyacrylamide gel under denaturing conditions, the low Mr heart enzyme shows a major band of Mr=73,000, comparable in size to the brain enzyme. The SDS-PAGE of the high and low Mr enzymes analyzed by immunoblotting with an antibody raised against low Mr brain endooligopeptidase A, showed a major antigen band corresponding to Mr=72,000. In addition, immunoblotting has also demonstrated that a monoclonal antibody antitubulinreacts with a polypeptide corresponding to Mr=50,000 present in the purified high Mr endooligopeptidase A. Both enzymes are activated by dithiothreitol and inhibited by thiol reagents, but are not affected by leupeptin, DFP or EDTA, thus indicating that they should be classified as nonlysosomal cysteinyl-endooligopeptidase A. Heart endooligopeptidase A Enkephalin-generating enzyme from heart BIOLOGICALLY active peptides are processed by limited proteolysis of larger precursor proteins known as prohormones (19). Observation of the amino acid sequence of the pro-hormones reveals that the structure of biologically active peptides within the precursor protein are, with few exceptions, flanked by paired basic residues suggesting a processing mechanism involving a proteolytic cleavage at this site (34). Such putative processing signals are present in pro-enkephalin (22) and pro-dynorphin (24) as well as in several naturally occurring enkephalin containing peptides derived from both precursors (37). Recently, enkephalins and a number of opioid peptides derived from pro-enkephalin and pro-dynorphin have been found in mammalian heart (23,39). To explain the presence of these opioid peptides in heart tissue an enkephalin generating system might also be present within cardiac cells. Among the enzymes that may participate on the formation of enkephalin is endooligopeptidase A (11,12). This enzyme isolated from the 25,000 g supernatant fraction of nervous tissue with Mr=71,000, is a cysteinyl endopeptidase with pH optimum of 7.2 and an isoelectric point of 5.2 (8, 13, 29). Characteristically, endooligopeptidase A is only effective on small polypeptides (9) with specificty towards the Phee-Ser~ bond of bradykinin (8) the ArgS-Arga bond of neurotensin (10) and the MetS-Arg6 of BAM-12P (11). More recently we reported on the ability of endooligopeptidase A to form enkephalins from a range of naturally occurring enkephalin containing peptides derived from both pro-enkephalin and pro-dynorphin (12). Enkephalin containing peptides ranging from 8 to 13 amino acids in size were shown to be good substrates for the enzyme, with 1Requests for reprints should be addressed to A. C. M. de Camargo. 945 946 CICILINI ET AL. ABBREVIATIONS Enk(Leu) Enk(Met) BAM-12P BAM-22P NT EDTA Tris DFP U HPLC SDS-PAGE Mr [LeuS]enkephalin [MetS]enkephalin Bovine adrenal medulla dodecapeptide Bovine adrenal medulla docosapeptide Neurotensin Ethylenediamine-tetraacetic acid Tris (hydroxymethyl)aminomethane Diisopropyl fluorphosphate Units of enzyme activity High performance liquid chromatography Sodium dodecyl sulfate polyacrylamide gel electrophoresis Relative molecular mass (molecular weight) enkephalin being the only product released from precursors, where this peptide is immediately followed by a pair of basic residues. Following the observation that heart is among the richest sources of endooligopeptidase A like enzyme (16) we report here on the isolation and characterization of two molecular weight forms of this enzyme in rabbit heart. EXPERIMENTALPROCEDURES Rabbit hearts were purchased from Granja Seleta (Itu, Sao Paulo, Brazil) and stored at -20°C. Synthetic peptides enk(Leu), enk(Met), BAM-12P, neurotensin and bradykinin were from Cambridge Research Biochemicals; dynorphin A,_8, dynorphin B, dynorphin A,-~7, neoendorphin (alpha), neoendorphin (beta), BAM-22P, were from Peninsula Laboratories Ltd.; <Glul-LeuZ-Tyr3-Glu4-Asn3-Lysn-ProT-Arg8 (neurotensin, 8) and Arg.~-Pro'°-Tyr"-Iler'-Leu'3 (neurotensin~_,:0 were obtained by incubation of neurotensin with rabbit brain endooligopeptidase A followed by purification over HPLC (10); Argl-Pro2-Pro3-Gly4-Phe 5 (bradykinin,j and Ser"-ProT-Phe~-Arg~ (bradykinin6_~) were synthesized by the solid-phase method and purified by counter-current distribution and ion-exchange chromatography by Professor A. C. M. Paiva and L. Juliano, Escola Paulista de Medicina, Sao Paulo, Brazil. Horseradish peroxidase immunoglobulin conjugates were from Dakko Corporation, Santa Barbara, CA. Bovine serum albumin, Aldolase, Blue Dextran 2000, Ribonuclease, Ovalbumin, Chymotrypsinogen and Sephacryl S-200 were from Pharmacia Fine Chemicals Inc. Diothiothreitol, aminopeptidase M, leupeptin, 5,5',-dithiobis (2nitrobenzoic) acid, sodium ethylenediamine tetraacetic acid, p-OH-mercuribenzoate and diisopropyl fluorphosphate were products of Sigma Chemical Co.; N-[1-(RS)-carboxy-2phenylethyl]-Ala-Ala-Phe-p-aminobenzoate and alpha-Nbenzoyl-Gly-Ala-Ala-Phe-p-aminobenzoatewere a generous gift of Dr. Marian Orlowski to O. Toffoletto and J. Rossier; Acrylamide N'-methylene-bis-acrylamide and acetonitrile were from Fluka AG, Buchs SG, Switzerland; DEAE-cellulose (Whatman DE-52) was obtained from Reeve Angel (London, United Kingdom). Aminex A-5 was from Bio-Rad Laboratories, Richmond, CA. Agarose was purchased from BDH Chemicals (Poole, United Kingdom). W-3 ion-exchange resin was from Beckman Instruments Co. and amino acid analyzer reagents were purchased from Pierce Chemical Co., Rockford, IL. Total cell extracts of epimastigote forms of Trypanosoma cruzi were used as a crude source of tubulin (38). Anti-veal skeletal muscle actin monospecific antibodies raised in rabbits were kindly provided by S. Avrameas, from Institute Pasteur, Paris. The monoclonal antibody against actin was purchased from Amersham, England. Enzyme Unit (U) This is defined as the amount of enzyme that hydrolyzes 1.0 ~mol of bradykinin per min at 37°C in 50 mM Tris-HCl, pH 7.5, containing 0.35 mM dithiothreitol and 10/zM substrate. Enzyme Extraction The enzyme was prepared at 4°C from 30 rabbit hearts homogenized in 1:3 weight:volume of l0 mM of Tris-HCl buffer pH 7.5, containing 0.25 M sucrose, for l rain at top speed in a 2 l Waring blender. The homogenate was centrifuged at 25,000×g for 60 rain and the precipitate discarded. Acid Precipitation The pH of the supernatant fraction of rabbit heart extract was adjusted to 5.0 by dropwise addition of 0.5 M acetic acid and maintained at 4°C for 15 min, the precipitate being discarded after centrifugation. The pH of the supernatant was then adjusted to 7.5 by addition of 0.5 M NaOH. DEAE-Celhdose Chromatography The DEAE-cellulose column (2.5×70 cm) was equilibrated with 50 mM Tris-HC1 buffer, pH 7.5, containing 30 mM NaC1. After sample application, the column was developed by stepwise elution with increasing NaCI concentrations. The column was operated at 220 mlhar at 4°C, and fractions of 9 ml were collected. Kininase activity (bradykinin inactivating activity) was determined by bioassay with isolated guinea pig ileum (7). The protein from the effluent was monitored by UV absorbance at 280 nm. Gel-Filtration on Sephacryl S-200 The column (2.5 × 110 cm) was equilibrated and developed at 4°C with 50 mM Tris-HCl buffer. The apparent Mr of the enzyme was estimated by the method of Andrews (2), using ribonuclease, chymotrypsinogen, bovine serum albumin and aldolase as Mr standards. Preparative Disc Electrophoresis on Polyacrylamide Gels Fractions DE-I, S-I, S-II and S-III (Figs. 1 and 2) were concentrated by ultrafiltration under reduced pressure and dialyzed at 4°C overnight against 50 mM Tris-HC1, pH 6.7, containing 20% glycerol. Polyacrylamide gel electrophoresis was carried out at 5°C, according to Ornstein-Davis method (18,32) on 6% acrylamide gels with bis-acrylamide: acrylarnide ratio 1:40. At the end of the run the gels were either stained for protein with Coomassie brilliant blue or assayed for enzyme activity as follows: the gel was out transversely into 40 slices and each piece was extracted with 0.5 ml of 50 mM Tris-HC1 buffer, pH 7.5, containing 30% glycerol at 4°C overnight. The material extracted from each slice was assayed for kininase activity. Preparation of Antibodies Anti-Endooligopeptidase A From Rat Brain Endooligopeptidase A was purified from rat brain minus cerebellum (Wistar; male; 400 g) essentially as previously described (13). The antibody against purified enzyme was E N K E P H A L I N G E N E R A T I O N AND ENDOOLIGOPEPTIDASE A ! E T 947 -15.0 20- E t..0- I oa (J z El n, O to 2.0- El 0.0 LJ -15.0 ~ ,_j E :D E W (j, E . ,_ > m t~. - o .7.s ~ "7 _J co Z < -7.5 E 1.0- >: I,-- to I.-U < < io - 00 --47 s~o 21s VOLUME 7.g 0.0 - , 0.0 1~0 VOLUME, mL L FIG. 1. DEAE-cellulose chromatography of the pH 5 supernatant fraction of the heart homogenate. The sample (1.87 g of protein and 26.6 U in 870 ml) was equilibrated with 50 mM Tris HCI buffer pH 7.5 containing 30 mM NaC1 and applied to the column (2.5×70 cm) equilibrated with the same buffer. The chromatography was carried out at 5°C under a flow rate of 220 ml/hr; fractions of 9 ml were collected. The elution of the proteins was achieved by stepwise increase of the NaCI concentration in the buffer as indicated by the arrows to 50, 70, 100 mM and 1.0 M, respectively. Kininase activity was determined by bioassay with isolated guinea pig ileum ( 0 - - 0 ) and protein was monitored by UV absorbance at 280 nm (--). The horizontal bars DE-I and DE-II indicate the pooled fractions under each peak displaying kininase activity. obtained by injecting subcutaneously 170 mU of enzyme (specific activity 1600 mU/mg) into male New Zealand rabbits using the scheme described in Camargo et al. (12) for the bovine enzyme. The IgG fraction of the antiserum was purified by DEAE-cellulose chromatography as the first protein peak emerging from the column at pH 8.6 buffered with 25 mM Tris-HC1 containing 35 mM NaCI (14). About 12 /xg of antibody thus purified is needed to inhibit 1.5 mU of rabbit heart endooligopeptidase A. SDS Gel Electrophoresis Lyophilized salt-free protein samples were taken up in 100/zl of 48 mM Tris-HCl buffer, pH 6.7, containing 1% SDS (w/v) and 6.5 mM of dithiothreitol, 0.5% bromophenol blue and 10% glycerol (v/v). After heating for 3 min at 100°C the samples were electrophoresed on a 7 to 15% linear gradient acrylamide gel slab according to the method of Laemmli (26), and stained as described above. Immunoblotting Immunoblotting was carried out essentially as described by Towbin et al. (36). Briefly, samples dissolved in boiling SDS-PAGE sample buffer containing 10% SDS and 10% 2-mercaptoethanol were electrophoresed in 6--16% polyacrylamide linear gradient microslab gels prepared according to Matsudaira and Burgess (28). After electrophoresis proteins were transferred to nitrocellulose sheets (Millipore, 0.45/xm pore size) for at least 2 hours at 200 mA. The trans- FIG. 2. Gel filtration on Sephacryl S-200 of the fraction DE-I. The pooled fractions under peak DE-I (Fig. 1) containing 11.6 U and 147 mg of protein were concentrated to 14 ml under reduced pressure at 5°C in an 8/32 Nojax Visking dialysis tube. This sample was applied to a column (2.5× 110 cm) equilibrated and developed at 5°C with 50 mM Tris-HCl buffer, pH 7.5 containing 100 mM NaCI, at a flow rate of 8.0 ml/hr. The horizontal bars S-I, S-II and S-Ill represent the pooled fractions containing 18%, 12% and 65% of the total effluent kininase activity, respectively. Kininase activity (O---O) and UV absorbance at 280 mm (--) were monitored as described in Fig. 1. ferred polypeptides and Mr markers were visualized by staining the sheets with 0.1% (w/v) Ponceau-S in 10% acetic acid. The sheets were subsequently soaked in blot buffer (150 mM NaCI, 1 mM EDTA, 30 mM Tris/HCl pH 7.3, 0.25% gelatin, 0.05% Tween 20 and 0.05% NAN3) for at least 30 minutes to block the remaining protein binding sites. After incubation with the rabbit antiserum against rat brain endooligopeptidase A diluted 1:50 and monoclonal antibody against tubulin YOL 1/34 (25) ascitic fluid-diluted 1:200 in blot buffer for 1 hr at room temperature, the sheets were subjected to three washes of 15 minutes each in blot buffer under constant motion. Bound immunoglobulins were visualized after incubation for 1 hr with the appropriate antiimmunoglobulin antibody coupled to horseradish peroxidase following three 10 min washes in PBS and reaction with diaminobenzidine (0.2 mg/ml) and H202 (5/zl of a 30% solution in 30 ml PBS). Determination of Products Derived From Bradykinin, Neurotensin and Enkephalin Containing Peptides Two methods were applied to determine the products formed when the peptides were incubated with heart peptidases. When bradykinin was used as substrate we applied the method described by Carvalho and Camargo (13) which consists of the application of the incubation mixture in an automatic amino acid analyzer (1) programmed to measure every arginine containing peptide derived from bradykinin. The products derived from neurotensin and enkephalin containing peptides were determined by reversed-phase HPLC on a Waters Associates system using a/~Bondaback C18 column (3.9×300 ram). The detailed experimental conditions are described elsewhere (12). CICILINI ET AL. 948 Enzyme Assays With Alpha-N-Benzoyl-Gly-Ala-Ala-Phe-pAmino-Benzoate The enzyme assays for high Mr and low Mr endooligopeptidase A were performed as described by Orlowski et al. (31) using alpha-N-benzoyl-Gly-Ala-Ala-Phep-aminobenzoate as substrate. In brief, the substrate (1 mM) was incubated in a final volume of 200/xl of 200 mM TrisHCI, pH 7.0, at 25°C, containing 0.2 mM dithiothreitol and 0.2 mU of purified high Mr or low Mr endooligopeptidase A. Incubations were performed at 37°C for 20 min and the reaction terminated by heat inactivation at 100°C for 2 min. Samples were then cooled on ice prior to a further 2 hr incubation at 37°C initiated by addition of 50 tzl of 5 mM dithiothreitol containing 20/zg aminopeptidase M. The amount of para-aminobenzoate released was determined by absorbance at 550 nm following diazotization as described (31). Protein and Peptide Concentration Measurements Protein was determined by the method of Bensadoun and Weinstein (4). The concentrations of low Mr endooligopeptidase A obtained from the acrylamide gel after electrophoresis and those of synthetic peptides were determined by amino acid analysis. Aliquots from purified enzyme and peptide solutions were lyophilized and subjected to acid hydrolysis as described by Simpson et al. (33). Hydrolyzates were analyzed using an automatic amino acid analyzer over a Beckman W-3 ion-exchange column according to Alonzo and Hirs (1). The amount of protein and peptides was calculated from the quantity of each amino acid residue determined by amino acid analysis. RESULTS Purification of Heart Endooligopeptidase A The pH 5 supernatant fraction of rabbit extract containing 1,970 mg of protein (specific activity 6.3 m U/mg) was applied on a DE-52 anion exchange column. The profile showed in Fig. 1 indicates the presence of two major components of bradykinin-inactivating activities eluted respectively at 50 and 70 mM NaC1. The solid bars at the bottom of Fig. 1 (DE-I and DE-II) indicate the pooled fractions. They correspond to 46% and 23% of the kininase activity introduced into the DE-52 column, respectively. The increase in specific activities was 5.7-fold for DE-I and 4.1-fold for DE-II. The fragments released from bradykinin indicated that the DE-I fraction contains endooligopeptidase A like activity since it releases stoichiometric amounts of bradykininl_~ and bradykinin6 9. The fragments released by the DE-II and III fractions revealed the predominance of exopeptidases acting on the carboxyl-terminus of bradykinin and was not further examined. Gel filtration of DE-I fraction on Sephacryl S-200 resulted in two peaks of kininase activity (Fig. 2). The high Mr peak was recovered in the void volume. The specific activity of the pooled fractions of the high Mr enzyme (solid bar, S-I in the bottom of Fig. 2) is 68.2 mU/mg protein. It corresponds to 7.4% of the total kininase activity present in the 25,000 × g × 60 min supernatant fraction of heart homogenate. The specific activity of the pooled fractions of the low Mr enzyme (solid bar, S-III in the bottom of Fig. 2) is 148.6 mU/mg protein yielding 14.4% of the total activity present in the 25,000xg supernatant fraction. The low Mr enzyme was estimated to have an apparent Mr=71,000 by gel filtration on Sephacryl S-200. The analysis of the fragments generated when bradykinin was incubated with the high Mr and low Mr enzymes indicates that both released stoichiometric amounts of bradykininl_,~ and bradykinin6 ~. The high Mr endooligopeptidase A enzyme loses 50% of kininase activity when stored at -20°C in the presence of 30% glycerol within the first 48 hours following gel filtration. Less than 10% of enzyme activity is found after one week storage period at -20°C in the presence or absence of 30% glycerol. On the other hand, the low Mr endooligopeptidase A can be stored for two months at -20°C in presence of 30% glycerol, without significant loss of activity. The S-I, S-I1, S-III and DE-I fractions were further purified by disc electrophoresis in polyacrylamide gels. Figure 3 shows the stained bands of proteins and the region where kininase activities were detected. For the PAGE of the high Mr enzyme the kininase activity is coincident with the major protein band. No kininase activity was detected in other segments of the gel. Attempts to extract the high Mr enzyme from the gel resulted in very low recovery of enzyme activity, usually less than 20%. Gel electrophoresis of the low Mr enzyme (Fig. 3) resulted in a single region of kininase activity where no visible band of protein could be observed. In contrast with the high Mr enzyme, the recovery of low Mr enzyme activity from the gel was about 80%. The yield of purified low Mr endooligopeptidase A recovered from each gel was 32 mU with specific activity of 3.8 U/mg. Figure 3 also shows the stained gels corresponding to fractions S-II and DE-I. Both exhibited two regions containing kininase activity which correspond to the activities found in the gels of fractions S-I and S-III. The enzymes obtained by preparative gel electrophoresis were analyzed by SDS-PAGE. Figure 4 shows the stained gels of high Mr and low Mr preparations of endooligopeptidase A after SDS-PAGE. A band corresponding to a polypeptide of Mr around 72,000 is observed in both high Mr and low Mr enzymes although several other bands are detected in the high Mr form of the enzyme by this procedure. Immunochemical Relationships of Heart Endooligopeptidase A, Brain Endooligopeptidase A and Cytoskeleton Proteins The immunoglobulin raised against rat brain endooligopeptidase A was tested against high Mr and low Mr heart endooligopeptidase A by immunoblotting after SDSPAGE. Low Mr and high Mr heart enzymes showed one major band of antigen with Mr=73,000 (Fig. 5, left) which is similar to Mr of rabbit brain endooligopeptidase A (13). The immunoblot of high Mr enzyme preparation exhibited two other bands of Mr=58,000 and 15,000 which could correspond to degradation products of low Mr form of endooligopeptidase A. It was also observed that high Mr enzyme preparation contained substantial amounts of tubulin whereas this protein was not detected in the low Mr enzyme (Fig. 5, right). In addition, the use of either a monoclonal or an antiactin polyclonal antibody did not disclose the existence of any immunoreactive analogue of the protein in both enzyme preparations (data not shown). Hydrolysis of Neurotensin, Enkephalin Containing Peptides and Alpha-N-Benzoyl-Gly-Ala-Ala-Phe-p-Aminobenzoate by Heart High Mr and Low Mr Endooligopeptidase A Due to the observation that brain endooligopeptidase A ENKEPHALIN GENERATION AND ENDOOLIGOPEPTIDASE DE-I. S-I ]- I]-- A 949 S-II S-Ill .41,-, .4,,,- 1.,4,,- ,41-- FIG. 3. Polyacrylamide gel electrophoresis of high Mr and low Mr heart endooligopeptidase A. The gels were loaded with about 40 mU of enzyme, corresponding to samples taken from fractions DE-I (Fig. 1), S-l, S-ll and S-Ill (Fig. 2), as indicated. Electrophoresis was carried out at 5°C for 17 hr with 0.6 mA/tube on 6% acrylamide gels. After the run the gels were stained with Coomassie blue and the position of the enzymatic activity determined on gels ran in parallel, indicated by brackets in the figure. The detailed procedures are described in the Experimental section. 950 CICIL1NI E T AL. $td High Mr LowHr the Experimental Procedures section. When neurotensin, BAM-12P and dynorphin B are incubated with either high Mr or low Mr endooligopeptidase A the only products formed are neurotensinl s and neurotensing_l.~ or the enkephalins with the respective complements BAM-12P6_~z and dynorphin B6-~3. Figure 6 illustrates the HPLC profile of the hydrolyzates of neurotensin, BAM-12P and dynorphin B by high Mr enzyme. The peptides eluted in each peak were collected and subjected to acid hydrolysis followed by amino acid analysis (data not shown) and correspond in a integral molar ratio to the following peptides: neurotensin~_8, neurotensing_~:+, neurotensin, BAM-12PHe, BAM-12P and enk(Met), dynorphin B6 1:~,dynorphin B and enk(Leu) (Fig. 6A, B and C). Both high Mr and low Mr endooligopeptidase A can also release enk(Leu) from neoendorphins (alpha and beta) and from dynorphin A~_s (Fig. 7). In analogy with brain endooligopeptidase A, the heart high Mr enzyme does not hydrolyze large enkephalin containing peptides such as BAM-22P and dynorphinl_~7. Similar results were obtained for low Mr heart endooligopeptidase A (data not shown). Table 1 shows the site of cleavage of the biologically active polypeptides studied here. The peptide benzoyl-Gly-AlaAla-Phe-p-aminobenzoate was not hydrolyzed by either of the high Mr and low Mr enzymes. Effects o f Various Substances on High Mr and Low Mr Endooligopeptidase A Activity When the enzyme assays are carried out in the presence of dithiothreitol and using dynorphin B as substrate, both high Mr and low Mr are activated (Table 3). The enzymes are fully inhibited by thiol reagents such as p-OHmercuribenzoate (1 mM) and 5,5'-dithiobis (2-nitrobenzoic) acid (3 raM) but not by 2 ~M leupeptin and by 10 mM DFP. Less specific thiol reagents such as Zn ++ and Cu ++ at 0.5 mM, also display a very strong inhibitory effect. Both low Mr and high Mr endooligopeptidase A are not significantly affected by EDTA or by N-[l-(RS)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, specific inhibitor of the metalloendopeptidase EC.3.4.24.15 (14). However, 11.8 /zg of anti-endooligopeptidase A immunoglobulins completely inhibit 1.3 mU of both low Mr and high Mr enzymes. DISCUSSION FIG. 4. SDS-PAGE of purified high Mr and low Mr heart endooligopeptidase A. The electrophoresis was performed on a 7-15% linear gradient slab gel and the protein bands detected by Coomassie blue staining. Both high Mr and low Mr endooligopeptidase A samples were obtained by preparative gel elec- trophoresis (Fig. 3). Mr markers ran in parallel correspond to the following proteins: bovine serum albumin, ovalbumin, chymotrypsinogen and ribonuclease A. The detailed procedures are described in the Experimental section. cleaves the Arg8-Arg9 bond of NT (10), the MeP-Arg 6 bond of BAM-12P (11) and the LeuS-Arg6 bond of dynorphin B (12) we investigated the ability of both high Mr and low Mr endooligopeptidase A from rabbit heart to hydrolyze these neuropeptides. Peptide substrates were incubated with each enzyme purified by gel electrophoresis and the reaction medium analyzed by reversed phase HPLC as described in In the present study, two endooligopeptidase A of different molecular weights were found in the cytosol of rabbit heart. Both low Mr and high Mr enzymes exhibited the same pattern of peptide bond cleavages of bradykinin, neurotensin and on enkephalin containing peptides as compared to the cysteinyl brain endooligopeptidase A. The presence of a high Mr endooligopeptidase A in nervous tissue has also been observed by Oliveira et al. (29). Inclusion of bradykinin in the incubation mixture resulted in a concentration dependent inhibition of enkephalin conversion by low Mr and high Mr heart endopeptidases (data not presented) thus indicating that the same enzyme cleaves both PheS-Ser6 bond of bradykinin and the MetS-Arg6 and Leu~-Arg6 bonds of enkephalin containing peptides to release enkephalins. It is also worth noting that both low Mr and high Mr heart endopeptidases cannot be considered metallo- or serine-endopeptidases because they were not significantly affected by EDTA or DFP. However, they were E N K E P H A L I N G E N E R A T I O N AND ENDOOLIGOPEPTIDASE A Low Tu 951 Low , 94-- 67-- 43-30-0 ~ ~ . 14_¸¸¸¸¸ FIG. 5. Immunoblotting analyses of high Mr and low Mr rabbit heart endooligopeptidase A using antibodies directed to rat brain endooligopeptidase A and to tubulin. Immunoblotting was carried out as described in the Experimental Procedures section. Left Panel: Samples of purified high Mr and low Mr enzymes as revealed by rabbit antibodies raised against purified rat brain endooligopeptidase A. Right Panel: Samples of Trypanosoma cruzi (Tu) whole cell extract and of both high Mr and low Mr enzyme preparations as revealed by monoclonal (YOL 1/34) antitubulin antibody. activated by dithiothreitol and fully inhibited by pOH-mercuribenzoate and by 5,5'-dithiobis (2-nitrobenzoic) acid which characteristically affects cysteinyl-proteinases. Endooligopeptidase A can also be distinguished from cathepsins because the enzyme is inactive at acid pH and does not hydrolyze denatured proteins (8,30). In addition, leupeptin, which at 2/xm concentration completely inhibits cathepsins [see (3)] showed no inhibitory effect towards endooligopeptidase A. Similarly to the cysteinyl brain endooligopeptidase A which cleaves only oligopeptides (9, 11, 12, 29) heart endopeptidases do not hydrolyze large opioid peptides. The resistance of large peptides to hydrolysis by heart enzymes seems to justify the general name of endooligopeptidase applied to these endopeptidases. The lack of susceptibility of large peptides and proteins to hydrolysis by low Mr and high Mr endopeptidases clearly discriminates between these enzymes and other multiple forms of intracellular proteinases such as Ca++-activated protease (41), Calpain (40) and multicatalytic proteinase (17). Recently Chu and Orlowski (15) have described a soluble metallo-endopeptidase EC.3.4.24.15 from rat brain with specificity similar to endooligopeptidase A. However, heart low Mr and high Mr endooligopeptidase A neither hydrolyze the EC.3.4.24.15 synthetic substrate nor are inhibited by the specific inhibitor of this enzyme (Table 2). Moreover, Chu and Oriowski (15) did not detect the presence of EC. 3.4.24.15 in the heart tissue where endooligopeptidase A was found in large quantities (16). In addition, we have demonstrated that brain endooligopeptidase A but not the EC.3.4.24.15 was able to generate enkephalin from dynorphinl_s enk(Met)-Arg-Arg-Val-NH2 (metorphamide) (35). Both enzymes were inhibited by the antiserum against rat brain endooligopeptidase A thus indicating that they contain at least some common epitopes. We have previously demonstrated that brain endooligopeptidase A is a protein of a single peptide chain of Mr=71,000 (13). A band of protein of similar Mr was also obtained for heart low Mr endooligopeptidase A. However, for high Mr enzyme the SDS gel electrophoresis showed several other bands besides the Mr 72,000 band, thus indicating that high Mr enzyme preparation contains other proteins which could be associated or contaminating the purified enzyme. Nevertheless, the lack of polypeptides of higher Mr suggests that high Mr form of endooligopeptidase A could consist of an associative form of low Mr enzyme with other cytosolic proteins. C I C I L I N I ET A L 952 A 1! ! OTT B OTT OTT 1; ! E L ,4" t,i / I ¢U • / j f / n,, I,-,. I..lJ o z I-- 0 u') m 4 w fj h rr' o o 11 O_ 5 10 15 5 10 TIME 15 5 10 15 (rain.) FIG. 6. Hydrolysis of NT (Panel A), BAM-12P (Panel B) and dynorphin B (Panel C) by high Mr endooligopeptidase A estimated by analysis over reversed phase HPLC. Peptides were separated by an initial isocratic elution in 0. I% H:~PO4, pH 2.7, followed by a 15 rain linear gradient up to 35% acetonitrile in 0. I% H:~PO~, pH 2.7, at a flow rate of 2 ml/min, and monitored by UV absorbance at 214 nm. The chromatograms show the hydrolysis of NT (12 nmol), BAM 12P (16 nmol) and dynorphin B (11 nmol) incubated in 1.0 ml of 50 nM Tris-HCl buffer, pH 7.5, containing 0.3 mM dithiothreitol at 37°C with 2.5 mU of high Mr endooligopeptidase A. The reaction was stopped by addition of 5 p.l of concentrated H:~PO~ to obtain 30 to 60% hydrolysis of substrate. The products separated by HPLC were collected and subjected to amino acid analysis after acid hydrolysis. The identification of each peak is indicated in the HPLC profiles. Dyn B, dynorphin B; DTT, dithiothreitol. 100- "S o C mr--_--, 50, , FIG. 7. Hydrolysis of enkephalin-containing peptides by purified endooligopeptidase A. Ten to fifteen nmol of each of the following synthetic substrates were incubated at 37°C with 1.3 mU of high Mr enzyme in 1.0 ml of 50 mM Tris-HCl buffer, pH 7.5, containing 0.3 mM dithiothreitol during 5, 10, 15, 30 and 45 min: neoendorphin (alpha) (A), neoendorphin (beta) (&), dynorphin A1_8 ([]), dynorphin AI-1r (~), dynorphin B (©), BAM 12P ( I ) and BAM 22P (0). The reaction was stopped by addition of 5/~1 of concentrated H 3 P O 4. The products of the enzymatic reaction were analyzed over reversed phase HPLC using an isocratic elution in 0.1% H 3 P O 4 , pH 2.7, containing 10% (v/v) acetonitrile followed by a linear gradient from 10% to 50% acetonitrile in 0.1% H3PO4, pH 2.7 at flow rate of 2 ml/min. Reaction products were monitored by absorbance at 214 rim. m i_ 0 G.) 04 w I 15 w I 30 TIME (rain) w I 45 953 E N K E P H A L I N G E N E R A T I O N AND ENDOOLIGOPEPTIDASE A TABLE 1 SITE OF CLEAVAGESOF PEPTIDESHYDROLYSEDBY HIGH Mr AND LOW Mr HEART ENDOOLIGOPEPTIDASEA T Bradykinin Neurotensin Arg- Pro- Pro- Gly- Phe- Ser- Pro- Phe- Arg <Glu-Leu-Tyr- Glu- Asn-Lys- Pro- Ar:Arg- Pro- Tyr- lie- Leu- t BAM-12P Tyr- Gly- Gly- Phe- Met-Arg- Arg- Val- Gly- Arg- Pro- Glu Dynorphin B Tyr- Gly- Gly- Phe- Le~Arg- Arg- Gin- Phe- Lys- Val- Thr- Arg Neoendorphin (alpha) Tyr- Gly- Gly- Phe- Le~Arg- Lys- Tyr- Pro- Neoendorphin (beta) Tyr- Gly- Gly- Phe- Le~Arg- Lys- Tyr Dynorphin A, Tyr- Gly- Gly- Phe- Leu~-Arg- Arg- lie The site of cleavage of peptide bond is indicated by an arrow. TABLE 2 EFFECT OF COMPOUNDSON THE FORMATIONOF ENK(LEU) FROMDYNORPHIN B BY HIGH Mr AND LOW Mr HEARTENDOOLIGOPEPTIDASEA Substance None Dithiothreitol 5,5'-Dithiobis (2-nitrobenzoic acid) p-OH-mercuribenzoate Leupeptin DFP EDTA CF-A-A-F-pAB ZnCI2 CuSO4 Anti-Brain-endooligopeptidase A-IgG Concentration (raM) Low Mr Rel. Activity (%) High Mr Rel. Activity (%) -0.2 0.4 0.8 1.5 3.0 0.5 1.0 0.002 10.0 1.0 2.0 0.3 0.5 0.5 11.8 tzg 100 110 140 130 25 0 15 0 100 84 100 85 90 0 0 0 100 120 130 110 35 0 20 0 100 78 80 65 100 0 0 0 The low Mr and high enzyme (1.3 mU) obtained by preparative gel electrophoresis was preincubated with the indicated compounds in 0.05 M Tris HCI buffer, pH 7.5, for 15 min at 4°C. Dynorphin B (11 nmol) was added and incubated for l0 min at 37°C. The reaction was stopped by addition of 5 /~l of concentrated HaPO4. Peptides were analysed by reversed phase HPLC according to the method described in the legend to Fig. 7. Somewhat indirect evidence that high Mr endooligopeptidase consists of an associative form of the low Mr species of the enzyme was obtained by the immunoblot analysis showed in Fig. 6. Both low Mr and high Mr enzyme react with antienzyme antibody producing a band of around 73,000 daltons which is similar to the Mr of the rabbit brain endooligopeptidase A 03). An apparent discrepancy emerges from the analysis of these results since the high Mr form of the enzyme was defined as the activity recovered in the exclusion volume of a liquid chromatography sizing step and both SDS-PAGE and the immunoblotting analysis indicated similar molecular weights for low Mr and high Mr forms of endooligopeptidase A. Since only major chemical modifications (such as carbohydrates) could yield differences in Mr on SDS-PAGE, we believe that the behaviour of high Mr endooligopeptidase A both in gel filtration and PAGE electrophoresis do reflect associative characteristics of this species. The same immunoblotting experiments also show antigens of Mr 58,000 and 15,000 in the high Mr preparation which could corre- 954 C I C I L I N I ET A L . spond to proteolytic fragments of the low Mr enzyme. This presumed susceptibility to proteolysis could also explain the low enzymatic stability of the high Mr enzyme described above. Other immunoblot studies also revealed the presence of tubulin in the high Mr enzyme preparation. This finding suggests that at least part of the endooligopeptidase A found in the 25,000xg supernatant fraction could be associated with cytoskeletal elements. It is a widespread observation to find cytosolic protein in association with the cytoplasmic matrix and the importance of such associations has been extensively studied in the enzymes of the intermediary metabolism (5, 6, 21, 27). One metabolic event which occurs during axonal transport is related to the processing of oxytocin and vasopressin in the secretory neurons of hypothalamus (20). 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